Method for detecting microorganisms

ABSTRACT

Methods for detecting microorganisms in a sample by binding detectable particles and fluorescent labelled ligands reactive to the microorganisms. The present invention also includes the use of multiple fluorochromes for the detection of microorganisms and is adaptable for use in flow cytometry.

TECHNICAL FIELD

The present invention relates to methods for detecting the presence ofmicroorganisms in a sample rising particles bearing a ligand reactive tothe microorganisms and fluorescent labelled ligands. The methods aresuitable for flow cytometric detection of microorganisms.

BACKGROUND ART

Testing samples for the presence of microorganisms. in particular humanpathogens. is an important part of monitoring samples includingbiological samples, foods, drinks, the environment and water supplies.In order to obtain immediate results testing often involves the directanalysis of samples for specific microorganisms. This can be labourintensive and routine for the technician involved. In particular. thereis an increasing need to monitor water supplies to ensure they meetstrict standards for human consumption. This often involves the testingof large volumes of water in order to detect relevant numbers ofmicrobial contaminants which is time consuming and expensive. Automatedmethods and apparatus are being developed to assist in the large scaletesting of samples for microbial contamination. The methods presently inuse are often insensitive and do not allow the identification ofspecific microorganisms present in the samples being tested.

Flow cytometric detection of specific microorganisms relies on labellingthe target organism with highly specific probes attached to fluorochromemolecules. To enable accurate detection. two or more differentfluorescent labels need to be attached to the target organism (Vesey etal. 1994A). The types of probes available for these techniques aremonoclonal and polyclonal antibodies, lectins and oligonucleotides. Therange of fltiorochromes that can be coupled to these probes is limited.For example, many flow cytometers utilise a 488 nm laser to illuminatethe sample, and accordingly, the choice of fluorochromes is limited tothose which can be excited at 488 nm for those machines.

For a flow cytometer to distinguish one fluorochrome from another, thefluorochromes must emit at different wavelengths. There are only threetypes of fluorochromes presently available that excite at 488 nm andemit at wavelengths different enough to be distinguished by flowcytometry: green fluorochromes such as fluorescein isothiocyanate(FITC); red fluorochromes such as phycoerythrin (PE) and tandemfluorochromes. Unfortunately, the tandem fluorochromes are often notbright enough to be used in many applications. Therefore, flow cytometryis often limited to the detection of two fluorochromes. In applicationssuch as the detection of specific microorganisms in a range of sampletypes, this poses a problem if there are only a small number of sitesavailable for recognition on the microorganism. The level of sensitivitythat can be achieved with two fluorochromes is often not good enough forthese applications.

The present inventors have developed methods of detecting microorganismsin a fluid sample utilising particles and fluorescent labelled ligandsreactive to microorganisms.

DISCLOSURE OF THE INVENTION

Accordingly. the present invention consists in a method of detecting thepresence of microorganisms of a predetermined type in a samplecontaining the microorganisms. the method comprising the steps of:

(a) treating the sample with at least one detectable particle, eachparticle bearing a ligand reactive to the microorganisms of thepredetermined type, the sample being treated for a period of timesufficient to allow the microorganisnms of the predetermined type in thesample to bind to the particle via the ligand;

(b) further treating the sample with at least one ligand labelled with afluorescent marker, the ligand being reactive to the microorganisms ofthe predetermined type, the sample being treated for a period of timesufficient to allow the at least one ligand to bind to themicroorganisms of the predetermined type; and

(c) analysing the sample so as to detect the presence of a particleassociated with one or more of the fluorescent markers, the ligandsbeing so selected that such an association is indicative of the presenceof microorganisms of the predetermined type in the sample.

In a preferred embodiment of the present invention the particle is afluorescent particle and more preferably a fluorescent latex bead. Thebeads preferably have a nominal diameter from 10 nanometres to 0.1millimetres. The beads are preferably detectable by virtue of beingfluorescently labelled. More than one type of particle can be used witheach type bearing a ligand reactive to the same or different type ofmicroorganism to be detected. It would, however, be within the scope ofthe invention to detect the bead by magnetism, by charge, by densitydifference or in any other suitable manner.

In a further preferred embodiment of the present invention the ligand isselected from the group consisting of antibody, lectin andoligonucleotide. Preferably, at least one of the ligands is a monoclonalantibody. When the particle is a fluorescent particle, the fluorescentmarkers attached to the at least one ligand have different fluorescentspectra to that of the fluorescent particle.

In a still further preferred embodiment. the analysing of the treatedsample is by flow cytometry. the microorganisms being detected by thepresence of fluorescence of the labelled ligand or in combination withthe size of the particle, or more preferably, fluorescence of both themarker and the particle. With regard to the detection of the size of theparticle, this includes either detecting the known size of the particleor detecting or measuring for an increased size caused by the binding ofmicroorganisms to the particle.

In a still further preferred embodiment of the present invention, theparticle is labelled with several ligands reactive to the same ordifferent microorganisms. Furthermore, several different particles canalso be used having the same or different ligands bound thereto. Forexample, in step (b) several different ligands reactive to the same ordifferent microorganisms but provided with different fluorescent markersare utilised to allow the possible detection of more than one type ofmicroorganism bound to the particle.

The method according to the present invention preferably uses one ormore fluorescent markers that are excited at 488 nm and emit atwavelengths ranging from green to infra-red. It will be appreciated byone skilled in the art that fluorescent markers that are excited atother wavelengths are also suitable for the present invention.

The present invention is suitable for detecting multiple forms of thesame species of microorganism or detecting several differentmicroorganisms from the same sample. The microorganisms bound to theparticle may be further treated or analysed after being detected by themethod of the present invention.

The number of particles used in the present methods will depend on thetype of particle, the type of sample being tested, and the number andtype of microorganisms in the sample. It will be appreciated that themicroorganism must come in contact with a particle to allow binding.Therefore, the number of particles should be in excess to the numbermicroorganisms in a given sample to ensure detection of themicroorganisms of interest. In order to assist in this regard usually atleast 10³ particles per ml, preferably between 10⁴ to 10⁷ particles perml are used. When a sample has a lot of particulate material presentthen usually a higher number of detectable particles is used in order toincrease the possibility that the microorganisms present in the samplewill come into contact with the particles and bind. The presentinvention has the advantage that the number of microorganisms in asample can also be estimated by adding a known number of detectableparticles to the sample and counting all of those particles to determinethe number that have bound microorganisms. Furthermore, by adding aknown number of particles to the sample it is also possible to confirmthat the sample was correctly analysed by enumerating the number ofparticles detected.

In order that the nature of the present invention may be more clearlyunderstood, preferred forms thereof will be described with reference tothe following examples and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows flow cytometry scatter plots representing Cryptosporidiumoocysts captured onto fluorescent beads and then bound with aFITC-conjugated Cryptosporidium-specific antibody; and

FIG. 2 shows flow cytometry scatter plots representing Adenoviruscaptured onto fluorescent beads and then bound with FITC-conjugatedAdenovirus-specific antibody.

MODES FOR CARRYING OUT THE INVENTION MATERIALS AND METHODS

Coating Beads with Antibody

TransFluorSphere 488/685 latex beads were coated as recommended byMolecular Probes (Eugene, USA) with antibody specific to themicroorganism of interest.

Antibody (2 mg) specific to either Cryptosporidium (Biox, Sydney),Adenovirus (Silenus, Melbourne) or Salmonella typhimurium (WellcomeDiagnostics) was dissolved in 1 ml of 50 mM Tris buffer (pH 8.4) andthen dialysed overnight at 4° C. against 50 mM MES buffer (pH 6.0). Theantibody was then mixed with 5 ml of 0.2% (w/v) 1μm latex beads(TransFluorSphere 488/685. Molecular Probes. Eugene USA) and incubatedat room temperature for 15 min before the addition of1-ethyl-3(3-dimethylaminopropyl)-carbodiimide (40 mg). The pH was thenadjusted to 6.5 by the addition of 0.1 M NaOH. After incubation at roomtemperature for 2 hours, glycine was added to give a final concentrationof 100 mM and the sample incubated for a further 30 min. The beads werethen pelleted by centrifuging (13000 g for 2 mil) and the pelletresuspended in 1% (w/v) bovine serum albumin (BSA) in phosphate bufferedsaline (pH 7.2). The washing procedure was repeated three times beforethe addition of 0.1%(w/v) sodium azide. The final sample volume was 4ml. Beads were sonicated for 30 min prior to use.

Using Beads to Label Cryptosporidium oocysts in Water Samples

River water samples were concentrated by calcium flocculation (Vesey etal. 1993). Portions (1 ml) of the concentrate were seeded withapproximately 1.000 oocysts. BSA was added to a concentration of 1%(w/v) prior to the addition of 20 μl of the crypto-antibody-coated beadsuspension. Samples were then incubated at room temperature on a rotarymixer for 30 min.

Oocysts were then labelled with a second fluorochrome. Monoclonalantibody, specific to Cryptosporidium oocysts walls, conjugated withfluorescein isothiocyanate (Cellabs Pty Ltd, Sydney, Australia) wasadded (0.5 ml) and the samples incubated at 37° C. for 20 min.

Using Beads to Label Salmonella

Salmonella typhimurium was cultured on MacConkey agar, fixed in 5% (v/v)formalin for 15 min and washed by centrifuging (13000 g for 10 min) andresuspending in PBS. An aliquot (100 μl) containing approximately 1×10⁶cells was mixed with 20 μl of bead suspension (coated with Salmonellaantibody) and then incubated on a rotary shaker for 30 min a roomtemperature. Salmonella cells attached to beads were labelled with asecond fluorochrome by incubating with rabbit anti-Salmonella antibody(Wellcome Diagnostics), washing by centrifuging at 13000 g for 30seconds. resuspending in a goat anti-rabbit7-amino-4-methylcoumarin-3-acetic acid (AMCA) (Dako, Glustop, Denmark)conjugated antibody and incubating for 10 min at 37° C. Samples wereexamined using epifluorescence microscopy. Samples were analysedimmediately.

Using Beads to Label Adenovirus

Adenovirus was cultured in human epithelial cells, harvested by freezethawing the cells and then purified from cell debris by centrifuging(13000 g for 2 min) and retaining the supernatant. The supernatant wasthen mixed with 20 μl of the bead suspension (coated with Adenovirusantibody) and then incubated on a rotary shaker for 30 min at roomtemperature. Adenovirus attached to beads were labelled with a secondfluorochrome by incubating with the same Adenovirus antibody conjugatedwith FITC for 20 min at 37° C.

Sample Analysis

Samples were analysed using a Becton Dickinson Facscan flow cytometer.The discriminator was set on red fluorescence (FL4) at a level slightlyless than the fluorescence of the beads. A region (R1) on a scatter plotof green fluorescence (FL1) verses side scatter (graph 1) was definedwhich enclosed the FITC-labelled oocysts. This region was then used togate a scatter plot of red fluorescence verses side scatter (graph 2). Aregion was defined on this second scatter plot which enclosed oocystsattached to beads. The same process was also used for analysis ofAdenovirus (FIG. 2).

Colour compensation was performed to separate the fluorescence of thebeads from the fluorescence of the labelled organism. Red fluorescencewas progressively subtracted from green fluorescence until a secondpopulation appeared on the green fluorescence verses side scatter graph.

RESULTS

Cryptosporidium

Analysis of the Cryptosporidium sample by flow cytometry resulted in adistinct population on graph 1 (FIG. 1). This population represents allbeads and was enclosed within a region (R1). Gating a graph of sidescatter verses FITC on the region R1 produced the scatter graph 2 (FIG.1). Two populations are observed on graph 2, a large population with alow green signal (spill over from high level of red fluorescence fromthe beads) which represent the beads and a smaller population with ahigh FITC signal which represent beads attached to FITC-labelledoocysts.

Adenovirus

Viruses could be detected on the flow cytometer when using the gatingand colour compensation procedures that were used for Cryptosporidium. Ascatter plot representing a large population of beads with low FITCfluorescence and a small population of beads with high FITC fluorescence(FIG. 2) was observed. This second population represents virusesattached to beads and labelled with FITC. The negative control did notcontain any beads with a high FITC signal.

Salmonella

Examination of the beads using epifluorescence microscopy revealed redfluorescing beads attached to blue (AMCA) fluorescing Salmoiiella cells.

The detection of specific or predetermined microorganisms with flowcytometry has the potential to replace existing methodologies for thedetection of microorganisins in samples ranging from clinical fluids,water, food and beverages. On way to enable simple and rapid flowcytometric detection of low numbers of microorganisms is to use at leasttwo different coloured fluorochromes for attached to the microorganisms.These fluorochromes are attached to the microorganism via highlyspecified ligands such as antibodies. This has been achieved previouslyby conjugating different coloured fluorochromes directly to antibodies(Vesey et al. 1994A).

The present inventors have shown that microorganisms can be detected byflow cytometer by attaching fluorescent beads to the microorganisms. Thepopulation representing oocysts attached to beads displayed in graph 2(FIG. 1) is an identifiable population totally clear from anyunassociated coloured bead or interfering noise. The populationrepresenting viruses attached to beads in FIG. 2 is also an identifiablepopulation.

The technique of using a fluorescent particle to tag a specific orpredetermined microorganism with a fluorescent label has severaladvantages over using only a fluorochrome-conjugated antibody. Firstly,only a single bead needs to be attached to the microorganism to achievedetectable fluorescence. To achieve the same level detection using onlya fluorochrome conjugated antibody requires thousands of antibodies tobe attached to the microorganism. These thousands of antibodies coverand mask available antigen sites. If only a single type of antigen isavailable on the surface of a microorganism, then it is not alwayspossible to label the surface of the organism with a second antibody.The present inventors have found that only one antigen is presented byCryptosporidium and therefore this organism is difficult to detect byprevious methods.

If the microorganism is labelled with an antibody coated fluorescentbead then there are many antigen sites still available on the surface ofthe organism for attaching an antibody conjugated to a fluorochrome.This technique enables two colour fluorescence labelling of amicroorganism with a single antibody as shown by the present inventors.

A further advantage of the fluorescent bead labelling technique is thatit enables the use of new fluorescence emission wavelengths. Untilrecently the number of different colours that can be detected by asingle laser flow cytometer has been limited to two. These are a greenfluorochrome such as FITC and a red fluorochrome such as PE. A thirdcolour is now possible using tandem fluorochromes such as PE/Texas redwhere the PE pumps the Texas red. These tandem fluorochromes however arenot bright enough for many applications. When attempting to detectmicroorganisms the fluorescence signals need to be very bright. Thefluorescent beads enable the use of a third and even a fourth verybright fluorescent signal. This is because beads with a range ofdifferent fluorescent emissions are available. Beads with emissions asfar into the infrared as 720 nm are available.

Examples of fluorescent beads and their production that are suitable foruse in the present invention can be found in U.S. Pat. No. 5,326,692.

The use of fluorescent beads as a label improves flow cytometricdetection. This is because the beads can be used as the size orfluorescence discriminator. The cytometer can be set so that it ignoresall other particles except for the beads. This overcomes coincidenceproblems due to the sample containing more particles than the cytometercan examine. It also means that a known number of particles need to beexamined for all samples.

The bead technology is highly applicable to the detection of bacteria.It enables multiple bright fluorescence signals to be achieved on thesurface of a range of bacteria. The production of antibodies to largegroups of bacteria (eg all gram negative bacteria) and then coatingbeads with these antibodies will allow the use of a single reagent for arange of microorganisms or their sub-types.

The application where this bead technology will have the most benefitswill be the flow cytometric detection of very small particles such asviruses. Coating beads with virus specific antibodies and reacting withsamples captures viruses onto the beads. The virus is then labelled witha second fluorochrome enabling detection. This is the first, simplevirus detection procedure that can be performed within minutes.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

REFERENCES

Vesey. G., Narai. J., Ashbolt. N., Williams. K. L. and Veal. D. 1994A.Detection of specific microorganisms in environmental samples using flowcytometry, p.489-522. In Methods in Cell Biology-Flow Cytometry SecondEdition. Academic Press Inc., New York.

Vesey. G., Hutton. P. E., Champion. A. C., Ashbolt. N. J., Williams. K.L., Warton. A. and Veal. D. A. 1994B. Application of flow cytometricmethods for the routine detection of Cryptosporidium and Giardia inwater. Cytometry, 16: 1-6.

Vesey. G., Slade, J. S., Byrne. M., Shepherd, K., and Fricker. C. R.,1993. A new method for the concentration of Cryptosporidium oocysts fromwater. J. Appl. Bact. 75.82-86.

What is claimed is:
 1. A method of detecting the presence and estimatingthe number of microorganisms of a predetermined type in a samplecontaining the microorganisms, the method comprising the steps of: (a)treating the sample with a detectable fluorescent particle, thefluorescent particle comprising an antibody which binds to themicroorganisms of the predetermined type, the sample being treated for aperiod of time sufficient to allow the microorganisms of thepredetermined type in the sample to bind to the fluorescent particle viathe antibody; (b) further treating the sample with an antibody labeledwith a fluorescent marker having a different fluorescent spectrum tothat of the fluorescent particle, the antibody being the antibody asused in step (a) which is capable of binding to the microorganisms ofthe predetermined type, the sample being treated for a period of timesufficient to allow the antibody to bind to the microorganisms of thepredetermined type; (c) analyzing the sample by flow cytometry so as todetect the presence of a fluorescent particle in combination with afluorescent marker, wherein such a combination with a fluorescent markeris indicative of the presence of microorganisms of the predeterminedtype in the sample; and (d) estimating the number of microorganisms ofthe predetermined type in the sample by measuring the intensity of thefluorescence of the fluorescent marker in combination with thefluorescent particle.
 2. The method according to claim 1 wherein thefluorescent particle is a fluorescent latex bead.
 3. The methodaccording to claim 2 wherein the fluorescent latex bead has a diameterfrom 10 nanometers to 0.1 millimeters.
 4. The method according to claim1 wherein the sample is treated with at least 10³ detectable fluorescentparticles per milliliter sample.
 5. The method according to claim 4wherein the sample is treated with between 10⁴ and 10⁷ detectablefluorescent particles per milliliter sample.
 6. The method according toclaim 1 wherein the microorganisms are selected from the groupconsisting of protozoa, bacteria, fungi and viruses.
 7. The method ofclaim 6 wherein the microorganisms are viruses.
 8. The method accordingto claim 1 wherein the antibody is a monoclonal antibody.
 9. The methodaccording to claim 1 wherein the fluorescent marker is excited at 488 nmand emits at wavelengths ranging from green to infra-red.
 10. The methodaccording to claim 1 wherein the microorganisms are detected by thepresence of fluorescence of the labeled antibody, by fluorescence of thelabeled antibody in combination with the size of the fluorescentparticle, or by fluorescence of both the antibody and the fluorescentparticle.
 11. The method according to claim 1 wherein the fluorescentparticle comprises at least two different antibodies.
 12. The methodaccording to claim 1 wherein step (b) further comprises at least twodifferent antibodies, each different antibody being labeled with adifferent fluorescent marker having a different fluorescence spectrum tothat of the fluorescent particle and the other labeled antibodies. 13.The method according to claim 1 wherein the detectable fluorescentparticle comprises at least two different antibodies, each antibodycapable of binding to a different predetermined type of microorganism,step (b) further comprises at least two different antibodies as in step(a), each different antibody being labelled with a different fluorescentmarker, having a different fluorescence spectrum to that of thefluorescent particle and the other labeled antibodies, and being capableof binding to a different type of microorganism so as to allow detectionof at least two types of microorganisms in the sample by analyzing forthe presence of at least two different fluorescent-labeled antibodies incombination with the fluorescent particle.
 14. The method according toclaim 1 wherein the number of microorganisms of a predetermined type inthe sample is estimated by adding to the sample a known number ofdetectible fluorescent particles and analyzing the fluorescent particlesin the sample for combination with one or more of the fluorescentmarkers and estimating the number of microorganisms in the sample fromthe number of detectible fluorescent particles associated with thefluorescent markers.